The examination of samples by means of microscopy is a broad technical field for which there are varied technical solutions. Starting from the standard light microscopy, widely different microscopy methods have evolved.
A standard field of use of light microscopy for examining biological preparations is fluorescence microscopy. In this process, particular dyes (so-called fluorophores) are used for the specific tagging of samples (e.g. of cell structures). The sample is illuminated by excitation radiation and the fluorescence radiation excited is recorded by suitable detectors. A dichroic beam splitter is usually provided in the light microscope in combination with block filters which split the fluorescence radiation from the excitation radiation and enable the fluorescence to be observed alone. Through this procedure, the light microscope can reveal individual, differently colored cell structures. Of course, different structures of a sample can also be simultaneously colored with different dyes attaching specifically to different structures of the sample. This procedure is called multiple luminescence. Samples which luminesce per se, and thus without added tagging substance, can also be surveyed.
Different approaches have recently been developed to obtain a resolution beyond the diffraction limit that follows from laws of physics. These microscopy methods are characterized in that they provide the user with a higher lateral optical resolution compared with a standard microscope. In this description, such microscopy methods are called high-resolution microscopy methods, as they achieve a resolution beyond the optical diffraction limit. Diffraction-limited microscopes, on the other hand, are called standard microscopes.
High-resolution widefield microscopy methods are disclosed in: T. Dertinger et al., “Fast, Background-Free, 3D Super-Resolution Optical Fluctuation Imaging (SOFI)”, PNAS (2009), pp. 22287-22292; “Achieving Increased Resolution and More Pixels with Superresolution Optical Fluctuation Imaging (SOFT)”, Opt. Express, 30.08.2010, 18(18): 18875-85, doi: 10.1364/IE.18.018875; and S. Geissbuehler et al., “Comparison Between SOFI and STORM”, Biomed. Opt. Express 2, 408-420 (2011). All of the foregoing references are hereby fully incorporated herein by reference. These methods use blinking characteristics of a fluorophore. If the fluorophores of a sample blink statistically and independently of one another, an imaging of the sample by suitable filtering with a so-called cumulant function achieves a significant increase in resolution beyond the physically specified optical resolution limit. An example of such a cumulant function is the second-order autocorrelation function. To produce a high-resolution image, a sample is excited and imaged in widefield. A sequence of frames is recorded and then combined using the cumulant function into one image which then has a higher resolution. This method is known in the art as the “SOFI” method, an abbreviation of the term “Super-Resolution Optical Fluctuation Imaging.”
In the SOFI method, a frame sequence with as many different blinking states as possible of all the fluorophores added to the sample or intrinsically present in the sample is required. At the same time, the camera used must be capable of recording this blinking over time and simultaneously offering a high spatial resolution. In the realization of the SOFI principle, as few fluorophores as possible should change their fluorescent state in a frame. The frame acquisition frequency must therefore be much higher than the blinking frequency of the fluorophores. In practice, due to the time response of available cameras, the range of possible fluorophores is therefore severely limited; only fluorophores for which the blinking rate is slow enough for the cameras employed, can be used. In the publication of Dertinger et al. cited above, so-called quantum dots are used which display a statistical blinking at almost all time scales. The vast majority of the fluorophores that have been developed in the state of the art for the most varied sample substances cannot be used in the SOFI method.